Transcriptomic and proteomic profiling of young and old mice in the Bleo model

The most preclinical used in vivo model to study lung fibrosis is the bleomycin-induced lung fibrosis model in 2-3-month-old mice. Although this model resembles key aspects of idiopathic pulmonary fibrosis (IPF), there are limitations in its predictability for the human disease. One of the main differences is the juvenile age of animals that are usually used in experiments, resembling humans of around 20 years. Because IPF patients are usually older than 60 years, aging appears to play an important role in the pathogenesis of lung fibrosis. Therefore, we here compared young (3 months) and old mice (21 months) 21 days after intratracheal bleomycin instillation. Analyzing lung transcriptomics (mRNAs & miRNAs) and proteomics, we found most pathways to be similarly regulated in young and old mice. However, old mice show an imbalanced protein homeostasis as well as an increased inflammatory state in the fibrotic phase compared to young mice. Comparisons with published human transcriptomic data sets (GSE47460, GSE32537 and GSE24206) revealed that the gene signature of old animals correlates significantly better with IPF patients as well as it turned human healthy individuals better into “IPF patients” using an approach termed predictive disease modelling. Comprising, young and old animals show similarly molecular hallmarks of IPF. Additionally, old mice resemble several features associated with IPF more closely compared to young animals. Overall design: Male mice were randomized before the study. Mice were instilled intratracheally (i.t.) under 3-4% isoflurane anesthesia once on day 0. Bleomycin administration was performed in a hanging position with a Vasofix-Braunüle 22G (B.Braun, Melsungen, Germany). Mice were instilled using 0.7 and 0.63 mg/kg of bleomycin sulphate (Merck, Darmstadt, Germany) in saline solution for the young and old mice, respectively. Control mice received saline solution only. Mice were analyzed 21 days after bleomycin administration.

目前用于肺纤维化研究的最常用临床前体内模型，为2~3月龄小鼠的博莱霉素诱导肺纤维化模型。尽管该模型复现了特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）的多项关键特征，但其对人类疾病的预测效能仍存在局限。其中一项核心差异在于实验所用动物均为幼年个体，对应人类约20岁的年龄段；而IPF患者的发病年龄通常超过60岁，提示衰老在肺纤维化的发病机制中发挥关键作用。因此，本研究对气管内滴注博莱霉素21天后的幼年（3月龄）与老年（21月龄）小鼠进行了对比分析。通过对肺组织转录组学（涵盖mRNA与microRNA，即miRNA）和蛋白质组学进行检测，我们发现多数信号通路在幼年与老年小鼠中呈现相似的调控模式。但相较于幼年小鼠，老年小鼠在纤维化阶段表现出蛋白质稳态失衡与炎症状态加剧的特征。将本研究数据与已发表的人类转录组数据集（GSE47460、GSE32537及GSE24206）进行比对后发现，老年动物的基因表达特征与IPF患者的相关性显著更高；同时，通过一种被称为预测疾病建模的分析方法，老年小鼠的特征可更精准地将健康人类个体归类为“IPF患者”。综上，幼年与老年小鼠均呈现IPF的相似分子特征；此外，相较于幼年动物，老年小鼠的特征与IPF相关的多项病理生理特征更为接近。整体实验设计：本研究在实验前对雄性小鼠进行随机分组。小鼠于第0天在3%~4%异氟烷麻醉下接受单次气管内（intratracheal, i.t.）滴注。给药时采用悬吊体位，使用22G Vasofix-Braunüle导管（德国贝朗医疗，梅尔松根）进行操作。幼年与老年小鼠分别给予0.7 mg/kg和0.63 mg/kg的硫酸博莱霉素（德国默克集团，达姆施塔特）生理盐水溶液；对照组小鼠仅给予生理盐水。博莱霉素给药21天后对小鼠进行样本分析。